Name: multiplexed immunofluorescence analysis and quantification of intratumoral pd-1+ tim-3+ cd8+ t cells
Uploaded: 2018-02-08T12:30:00
Description: Watch this Scientific Journal Video about Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells at JoVE.com

Dette arbeidet ble støttet av tilskudd fra Institut nasjonale du kreft (INCA) (ET) Ligue contre le kreft (ET), Université Sorbonne Paris Cité (ET), ANR (Selectimmunco) (ET), Labex Immuno-onkologi (ET), SIRIC CARPEM (CG, ET). EdG ble finansiert av et fellesskap av Fondation bue. EV og CD ble finansiert av et fellesskap av APHP (bourse année recherche). Les er finansiert av et fellesskap av Université Sorbonne Paris Cité (contrat doktorgrad). Forfatterne takker Bristol Myers Squibb for sin finansiering i dette prosjektet. Forfatterne takker Institutt for patologi på Hopital Européen Georges Pompidou og Necker (Laurianne Chambolle, Elodie Michel og Gisèle Legall). Forfatterne takker Histology plattformen for PARCC, Hopital Européen Georges Pompidou (Corinne Lesaffre).

Denne studien ble gjennomført i henhold til erklæringen i Helsinki og ble godkjent av den lokale etikk (CPP Ile de France nr. 2012-05-04). Informert samtykke ble innhentet fra deltaker inkludert i kohortene.
1. vev materiale
<ol><li>Samle RCC vevsprøver på dagen. Håndtere kirurgiske prøven ved romtemperatur (patologi avdeling).</li>
	<li>Samle et svulst utvalg av ca 0,5 cm x 0,5 cm x 0,5 cm størrelse i en tørr rør. Fest fryse i flytende nitrogen og lagre på-80 ° C.</li>
	<li>Coat ...

<ol>
	<li>Borghaei, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nivolumab+versus+Docetaxel+in+Advanced+Nonsquamous+Non-Small-Cell+Lung+Cancer.">Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer.</a> N Engl J Med. 373 (17), 1627-1639 (2015).</li><li>Granier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunotherapy:+Rational+and+recent+breakthroughs.">Cancer immunotherapy: Rational and recent breakthroughs.</a> Rev Med Interne. 37 (10), 694-700 (2016).</li><li>Motzer, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nivolumab+for+Metastatic+Renal+Cell+Carcinoma:+Results+of+a+Randomized+Phase+II+Trial.">Nivolumab for Metastatic Renal Cell Carcinoma: Results of a Randomized Phase II Trial.</a> J Clin Oncol. 33 (13), 1430-1437 (2015).</li><li>Robert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nivolumab+in+previously+untreated+melanoma+without+BRAF+mutation.">Nivolumab in previously untreated melanoma without BRAF mutation.</a> N Engl J Med. 372 (4), 320-330 (2015).</li><li>Robert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ipilimumab+plus+dacarbazine+for+previously+untreated+metastatic+melanoma.">Ipilimumab plus dacarbazine for previously untreated metastatic melanoma.</a> N Engl J Med. 364 (26), 2517-2526 (2011).</li><li>Rosenberg, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atezolizumab+in+patients+with+locally+advanced+and+metastatic+urothelial+carcinoma+who+have+progressed+following+treatment+with+platinum-based+chemotherapy:+a+single-arm,+multicentre,+phase+2+trial.">Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial.</a> Lancet. 387 (10031), 1909-1920 (2016).</li><li>Schadendorf, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pooled+Analysis+of+Long-Term+Survival+Data+From+Phase+II+and+Phase+III+Trials+of+Ipilimumab+in+Unresectable+or+Metastatic+Melanoma.">Pooled Analysis of Long-Term Survival Data From Phase II and Phase III Trials of Ipilimumab in Unresectable or Metastatic Melanoma.</a> J Clin Oncol. 33 (17), 1889-1894 (2015).</li><li>Ribas, A., Hu-Lieskovan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+PD-L1+positive+or+negative+mean?.">What does PD-L1 positive or negative mean?.</a> J Exp Med. 213 (13), 2835-2840 (2016).</li><li>Rizvi, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunology.+Mutational+landscape+determines+sensitivity+to+PD-1+blockade+in+non-small+cell+lung+cancer.">Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer.</a> Science. 348 (6230), 124-128 (2015).</li><li>Roussel, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+biomarkers+defined+by+multiparametric+immunofluorescence+analysis+identify+ALK-positive+adenocarcinoma+as+a+potential+target+for+immunotherapy.">Composite biomarkers defined by multiparametric immunofluorescence analysis identify ALK-positive adenocarcinoma as a potential target for immunotherapy.</a> OncoImmunology. 6 (4), e1286437 (2017).</li><li>Pages, F., Granier, C., Kirilovsky, A., Elsissy, C., Tartour, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarqueurs+pr&#233;dictifs+de+r&#233;ponse+aux+traitements+bloquant+les+voies+de+costimulation+inhibitrices.">Biomarqueurs pr&#233;dictifs de r&#233;ponse aux traitements bloquant les voies de costimulation inhibitrices.</a> Bull Cancer. 103, S151-S159 (2016).</li><li>Tumeh, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1+blockade+induces+responses+by+inhibiting+adaptive+immune+resistance.">PD-1 blockade induces responses by inhibiting adaptive immune resistance.</a> Nature. 515 (7528), 568-571 (2014).</li><li>Fourcade, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+Tim-3+and+PD-1+expression+is+associated+with+tumor+antigen-specific+CD8++T+cell+dysfunction+in+melanoma+patients.">Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients.</a> J Exp Med. 207 (10), 2175-2186 (2010).</li><li>Koyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+resistance+to+therapeutic+PD-1+blockade+is+associated+with+upregulation+of+alternative+immune+checkpoints.">Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints.</a> Nat Commun. 7, 10501 (2016).</li><li>Wherry, E. J., Kurachi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+insights+into+T+cell+exhaustion.">Molecular and cellular insights into T cell exhaustion.</a> Nat Rev Immunol. 15 (8), 486-499 (2015).</li><li>Cai, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tim-3+expression+represents+dysfunctional+tumor+infiltrating+T+cells+in+renal+cell+carcinoma.">Tim-3 expression represents dysfunctional tumor infiltrating T cells in renal cell carcinoma.</a> World J Urol. 34 (4), 561-567 (2016).</li><li>Sakuishi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Tim-3+and+PD-1+pathways+to+reverse+T+cell+exhaustion+and+restore+anti-tumor+immunity.">Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity.</a> J Exp Med. 207 (10), 2187-2194 (2010).</li><li>Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+immunohistochemistry,+imaging,+and+quantitation:+a+review,+with+an+assessment+of+Tyramide+signal+amplification,+multispectral+imaging+and+multiplex+analysis.">Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.</a> Methods. 70 (1), 46-58 (2014).</li><li>Bethmann, D., Feng, Z., Fox, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprofiling+as+a+predictor+of+patient's+response+to+cancer+therapy-promises+and+challenges.">Immunoprofiling as a predictor of patient's response to cancer therapy-promises and challenges.</a> Curr Opin Immunol. 45, 60-72 (2017).</li><li>Badoual, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1-expressing+tumor-infiltrating+T+cells+are+a+favorable+prognostic+biomarker+in+HPV-associated+head+and+neck+cancer.">PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer.</a> Cancer Res. 73 (1), 128-138 (2013).</li><li>Nizard, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+resident+memory+T+cells+enhances+the+efficacy+of+cancer+vaccine.">Induction of resident memory T cells enhances the efficacy of cancer vaccine.</a> Nat Commun. 8, 15221 (2017).</li><li>Nghiem, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1+Blockade+with+Pembrolizumab+in+Advanced+Merkel-Cell+Carcinoma.">PD-1 Blockade with Pembrolizumab in Advanced Merkel-Cell Carcinoma.</a> N Engl J Med. 374 (26), 2542-2552 (2016).</li><li>Roussel, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+biomarkers+defined+by+multiparametric+immunofluorescence+analysis+identify+ALK-positive+adenocarcinoma+as+a+potential+target+for+immunotherapy.">Composite biomarkers defined by multiparametric immunofluorescence analysis identify ALK-positive adenocarcinoma as a potential target for immunotherapy.</a> Oncoimmunology. (4), (2017).</li><li>Woods, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mismatch+in+epitope+specificities+between+IFNgamma+inflamed+and+uninflamed+conditions+leads+to+escape+from+T+lymphocyte+killing+in+melanoma.">Mismatch in epitope specificities between IFNgamma inflamed and uninflamed conditions leads to escape from T lymphocyte killing in melanoma.</a> J Immunother Cancer. 4, 10 (2016).</li><li>Granier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tim-3+Expression+on+Tumor-Infiltrating+PD-1+CD8++T+Cells+Correlates+with+Poor+Clinical+Outcome+in+Renal+Cell+Carcinoma.">Tim-3 Expression on Tumor-Infiltrating PD-1+CD8+ T Cells Correlates with Poor Clinical Outcome in Renal Cell Carcinoma.</a> Cancer Res. 77 (5), 1075-1082 (2017).</li><li>Feng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+Imaging+of+T+and+B+Cells+in+Murine+Spleen+and+Tumor.">Multispectral Imaging of T and B Cells in Murine Spleen and Tumor.</a> J Immunol. 196 (9), 3943-3950 (2016).</li><li>Feng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+imaging+of+formalin-fixed+tissue+predicts+ability+to+generate+tumor-infiltrating+lymphocytes+from+melanoma.">Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma.</a> J Immunother Cancer. 3, 47 (2015).</li><li>Fridman, W. H., Pages, F., Sautes-Fridman, C., Galon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+contexture+in+human+tumours:+impact+on+clinical+outcome.">The immune contexture in human tumours: impact on clinical outcome.</a> Nat Rev Cancer. 12 (4), 298-306 (2012).</li><li>Nizard, M., Roussel, H., Tartour, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+Memory+T+Cells+as+Surrogate+Markers+of+the+Efficacy+of+Cancer+Vaccines.">Resident Memory T Cells as Surrogate Markers of the Efficacy of Cancer Vaccines.</a> Clin Cancer Res. 22 (3), 530-532 (2016).</li><li>Sandoval, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+imprinting+of+vaccine-induced+CD8(+)+T+cells+is+crucial+to+inhibit+the+growth+of+mucosal+tumors.">Mucosal imprinting of vaccine-induced CD8(+) T cells is crucial to inhibit the growth of mucosal tumors.</a> Sci Transl Med. 5 (172), 172ra120 (2013).</li><li>Carstens, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+computation+of+intratumoral+T+cells+correlates+with+survival+of+patients+with+pancreatic+cancer.">Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer.</a> Nat Commun. 8, 15095 (2017).</li></ol>

Endringer og feilsøking:
Vev kvaliteten er en viktig parameter; Det kan enkelt kontrolleres ved hematoxylin og eosin farge.
En fordel med teknikken er muligheten for multiplex stainings, men for å unngå fluorophore smitteeffekter, anbefales det for å velge utslipp bølgelengder med delta 10 nm minimum. Her, fordi vi hadde 4 stainings inkludert DAPI, valgte vi å fordele bølgelengdene (DAPI: 460 nm, AF488: 519 nm, Cyanine 3:570 nm, Cyanine 5:670...

I de siste årene, har immunterapi framstått som en meget lovende behandling for mange typer kreft, inkludert RCC. Spesielt immunterapi basert på hemming av hemmende sjekkpunkter som PD-1 og CTLA-4 har blitt rapportert å være klinisk effektiv1,2,3,4,5, 6. monoklonale antistoffer mot CTLA-4, PD-1 eller Program død Ligand 1 (PD-L1) er allerede godkjent i flere typer kreft og føre langvarig kliniske tiltak i mer enn 20% av pasientene7. Likevel, ikke alle pasienter er respondere, behandling er høy og disse behandlingene er giftige, fører til potensielle alvorlig autoimmun-lignende bivirkninger. Derfor er nåværende utfordringen å identifisere prediktiv indikatorer som de nye immunotherapies. Frekvensen av mutasjoner i svulsten, uttrykk for PD-L1 eller nivåer av intratumoral CD8+ T celle infiltrasjon har blitt rapportert å korrelere med klinisk respons. Denne tilknytningen er imidlertid fortsatt svak anbefaler bruk av disse kliniske biomarkers i klinisk praksis bortsett fra kameraten testen for PD-L1 før administrasjonen av Pembrolizumab i ikke-småcellet lungekreft kreft (NSCLC) pasienter8 ,9,1011,12. Det har blitt demonstrert at co uttrykk for mange hemmende reseptorer som PD-1, Tim-3, Lag-3, og CTLA-4, induserer en celle utmattelse fenotypen og motstand mot terapi13,14,15. Siden perifert blod ikke er representative for svulst microenvironment, er det stor interesse å analysere fenotypiske funksjonene i cellene i situ. PD-1 og Tim-3 co uttrykke T-celler er kjent for å være funksjonelt nedsatt celler i flere sammenhenger13,16,17. I denne studien, prognostiske virkningen av co uttrykk for to hemmende receptors PD-1 og Tim-3 på CD8+ T celler ble vurdert.
Opp til nå, studere co uttrykk for flere indikatorer på svulst infiltrere lymfocytter (TILs) er hovedsakelig utført av flyt cytometri analyse, noe som gjør det nødvendig å arbeide med fersk svulster og derfor utelukker retrospektiv analyser. Med konvensjonelle i situ flekker, eneste flekker samtidig kan utføres, og karakterisering av hvilken celle som co uttrykker markørene er ikke mulig. For eksempel er PD-L1 uttrykt ved mange celletyper av tumoral microenvironment, gjør det vanskelig å definere ved konvensjonelle immunohistochemistry analyse hvilke celler uttrykke PD-L1 er mer relevante for correlative studier. I dette arbeidet, vi utviklet en nyskapende i situ multiparametric immunofluorescence metode med datamaskin-telling for å sammenligne co uttrykket av PD-1 og Tim-3 av svulst infiltrere CD8+ T-celler med kliniske utfall i RCC. Denne teknikken har flere fordeler, inkludert muligheten til å analysere på en enkelt celle og flere markører samtidig med et multispectral kamera som kan fange begrenset intervaller > 10 nm gjennom krystall filtrerer18. Videre er fremgangsmåten automatisk som muliggjør en mellom operatører reproduserbarhet og en forkortet analyse forhold til manuelle teknikker19. I feltet kreft rapportert flere studier overbevise flere stainings av immun molekyler som PD-1, PD-L1 og CD8 Merkel celle carcinoma, lungekreft og hode og nakke kreft20,21,22, 23. de automatiserte celletall er mulig med opplæring av brukeren (phenotyping trinn). Fluorescensen måles i de ulike celle avdelinger (kjerner, cytoplasma og membran).
Her, forskjellige undergrupper av svulst-infiltrere CD8+ T celler uttrykke PD-1 og/eller Tim-3 i en stor kohort av RCC ble regnet og resultatene var korrelert med klinisk tyngdekraften score og overlevelse parametere. Det var også mulig å analysere membran fluorescens intensitet på mobilnettet oppløsningen mener fluorescens intensitet (MFI) data som i cytometri. Så vidt vi vet, er dette de første studien rapportering prognostiske resultatene bruker denne multispectral tenkelig basert teller teknikken.

<table>
	<tbody>
		<tr>
			<td>Vectra 3 Automated Quantitative Pathology Imaging&#160;</td>
			<td>Perkin Elmer</td>
			<td>CLS142338</td>
			<td></td>
		</tr>
		<tr>
			<td>inForm cell analysis 2.1.</td>
			<td>Perkin Elmer</td>
			<td>CLS135781</td>
			<td></td>
		</tr>
		<tr>
			<td>R software</td>
			<td>https://www.r-project.org</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dakopen delimiting pen</td>
			<td>Dako</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Buffer Salin TBS Tablets</td>
			<td>Takara, Bio Inc.</td>
			<td>TAKT9141Z</td>
			<td>pH7.6 100 tablet</td>
		</tr>
		<tr>
			<td>Tris Buffer Salin Tween 20 TBS(+Tween20)</td>
			<td>Takara, Bio Inc.</td>
			<td>TAKT9142Z</td>
			<td>pH 7.6 100 tablets</td>
		</tr>
		<tr>
			<td>Biotin blocking system</td>
			<td>Dako</td>
			<td>X0590</td>
			<td>Avidin 0.1% and Biotin 0.01%</td>
		</tr>
		<tr>
			<td>normal donkey serum</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-001</td>
			<td>5% vol./vol. concentration</td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma-aldrich</td>
			<td>F6057</td>
			<td>1.5 &#181;g/mL concentration&#160;</td>
		</tr>
		<tr>
			<td>Knittel glass coverslip</td>
			<td>Knittel&#160;Gl&#228;ser,</td>
			<td>100039</td>
			<td>24x60 mm 100 cover slips</td>
		</tr>
		<tr>
			<td>Rabbit anti-CD8 Clone P17-V</td>
			<td>novus</td>
			<td>NBP1-79055</td>
			<td>use at 4&#181;g/mL</td>
		</tr>
		<tr>
			<td>Mouse anti-PD-1 Clone NAT</td>
			<td>abcam</td>
			<td>ab52587</td>
			<td>use at 2 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Goat anti-Tim-3</td>
			<td>R&#38;D</td>
			<td>AF2365</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Rabbit anti-PD-L1 Clone SP142</td>
			<td>Roche</td>
			<td>7309457001</td>
			<td>use at 1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Mouse AF647 labeled pan- Keratin Clone C11&#160;</td>
			<td>Cell Signalling</td>
			<td>4528</td>
			<td>use at 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Goat anti-human gal9&#160;</td>
			<td>R&#38;D</td>
			<td>AF2045</td>
			<td>use at 0.3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Cyan 5 conjugated donkey anti-rabbit&#160;</td>
			<td>Jackson Immunoresearch</td>
			<td>711-175-152</td>
			<td>use at 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Biotinylated F(ab&#8217;2) donkey anti-mouse IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>715-066-150</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Alexa Fluor488 conjugated donkey anti-goat IgG</td>
			<td>abcam</td>
			<td>ab150133</td>
			<td>use at 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Cy3 labeled streptavidin&#160;</td>
			<td>Amersham</td>
			<td>PA43001</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>negative control mouse IgG1</td>
			<td>Dako</td>
			<td>X0931</td>
			<td>use at 2 &#181;g/mL</td>
		</tr>
		<tr>
			<td>IgG from goat serum</td>
			<td>Sigma-aldrich</td>
			<td>I5256</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>IgG from rabbit serum</td>
			<td>Sigma-aldrich</td>
			<td>I5006</td>
			<td>use at 4&#181;g/mL</td>
		</tr>
	</tbody>
</table>

multiplexed immunofluorescence analysis and quantification of intratumoral pd-1+ tim-3+ cd8+ t cells

Immunceller er viktige komponenter i svulst microenvironment og påvirke tumor vekst og utviklingen i alle stadier av kreft. Spesielt er det nå godt etablert at det immun infiltrere i menneskelig svulster kan sammenligne med prognose og respons på behandling. Analyse av den immun infiltrere i svulst microenvironment har blitt en stor utfordring for klassifisering av pasienter og respons på behandling.
Co uttrykk for hemmende reseptorer som programmet celle død Protein 1 (PD1, også kjent som CD279), cytotoksisk T lymfocytt forbundet Protein 4 (CTLA-4), T-celle immunglobulin og Mucin som inneholder Protein-3 (Tim-3, også kjent som CD366) og lymfocytter Aktivisering genet 3 (Lag-3, også kjent som CD223), er et kjennetegn på T celle utmattelse. Vi utviklet en multiparametric i situ immunofluorescence flekker og kvantifisere på cellenivå co uttrykket av disse hemmende reseptorer. På en retrospektiv rekke frossent av nyre celle kreftsvulster (RCC), bruke en fluorescens multispectral imaging teknologi kombinert med en analyseprogramvare, ble det funnet co uttrykket av PD-1 og Tim-3 på svulst infiltrere CD8+ T celler er korrelert med en dårlig prognose i RCC. Vi vet, dette representerer den første studien viser at dette automatisert multiplex i situ teknologi kan ha noen klinisk relevans.

Bruke den generelle protokollen beskrevet ovenfor, vi som mål å kvantifisere intratumoral CD8+ T celler uttrykke co hemmende receptors PD-1 og Tim-3 i frosne vev fra pasienter med RCC, og å korrelere resultatene med kliniske utfall25.
Optimalisering av den CD8/PD-1/Tim-3 flekker:
Ulike arter av antistoffer (mus, ka...

Watch this Scientific Journal Video about Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells at JoVE.com

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Multiplekset Immunofluorescence analyse og måling av Intratumoral PD-1+ Tim-3+ CD8+ T celler

Studere svulst microenvironment kan identifisere prognostiske eller prediktiv biomarkers av kliniske svar immunterapi. Presenteres her, er en innovativ metode basert på i situ fluorescens multispectral imaging for å analysere og telle automatisk ulike subpopulasjoner av CD8+ T celler. Denne reproduserbare og pålitelig teknikken er egnet for store kohort analyser.

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it ...

Research

JoVE Journal

Cancer Research

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex Vivo Avbildning av bosatt CD8 T-lymfocytter i menneskelig lunge svulst skiver bruker AC Confocal mikroskopi

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Sanntid påvisning av i Vitro svulst celle apoptose indusert av CD8+ T celler å studere immun undertrykkende funksjoner av svulst-infiltrere myeloide celler

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Patologisk analyse av lungemetastase etter lateral hale-vene injeksjon av tumorceller

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

Evaluering av celledød ved hjelp av cellefri supernatant av probiotika i tredimensjonale sfæroidkulturer av kolorektale kreftceller

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

Immunofluorescens Avbildning av DNA-skade og reparasjon av foci i humane tykktarmskreftceller

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Tumortransplantasjon for å vurdere dynamikken i tumorinfiltrerende CD8 + T-celler hos mus

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Expansion and Enrichment of Gamma-Delta ÃƒÅ½Ã‚Â³ÃƒÅ½Ã‚Â´ T Cells from Apheresed Human Product

Utvidelse og berikelse av Gamma-Delta (γδ) T-celler fra det afærerte menneskelige produktet

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed Barcoding bildeanalyse for immunprofilering og romlig kartlegging karakterisering i enkeltcelleanalyse av parafinvevsprøver

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment (Video) | JoVE 

Multiplex immunfluorescens kombinert med romlig bildeanalyse for klinisk og biologisk vurdering av tumormikromiljøet

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

Flow Analysis of PDL1-Vaxx Antibody Blockade Using Magnetic Fluorescent Beads

Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction

Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction (Video) | JoVE

Magnetisk fluorescerende perlebasert dual-reporter strømningsanalyse av PDL1-vaxx peptidvaksineindusert antistoffblokade av PD-1 / PD-L1-interaksjonen

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer ...